The electronic industry shows a constant effort in obtaining high density wiring and circuits. One key issue in reaching this goal is to make the individual wires as small as possible. One approach known in the prior art is the metallisation of nucleic acids which, once metallised, serves as an electrically conducting wire.
In addition, the “metalation” of nucleic acids is known, which refers to the process of direct bonding between a metal atom and a site within the nucleic acid, especially to the N-7 atoms of the purine nucleotides (G and A). Such reactions have been widely studied because of their relevance to the mechanisms of anti-cancer drugs, mostly Pt (II) or Pt (IV) complexes (“platination”). Other metal complexes exhibiting this behavior include the complexes of Pd, Ru, Au, Rh. The complex requires at least one “labile” ligand as a “leaving group” in order to bind in this manner.
Further, nucleic acid binding agents have been widely studied as anti-cancer drugs. Non-covalent binding agents include “intercalators” and “groove binders”. Agents that bind covalently are generally called “alkylators”. Many examples of each class of agents are known, as well as molecules with combined functions. Selectivity towards specific base pair combinations or sequences or other “recognition sites” is tuneable to a high degree (e.g. “drug targeting”).
WO 99/04440, published on Jan. 28, 1999, describes a three-step process for the metallisation of DNA. First, silver ions (Ag+) are localized along the DNA through Ag+/Na+ ion-exchange and formation of complexes between the Ag+ and the DNA nucleotide bases. The silver ion/DNA complex is then reduced using a basic hydroquinone solution to form silver nanoparticles bound to the DNA skeleton. The silver nanoparticles are subsequently “developed” using an acidic solution of hydroquinone and Ag+ under low light conditions, similar to the standard photographic procedure. This process produces silver wires with a width of about 100 nm with differential resistance of about 10 MΩ.
Further, Pompe et al. (Pompe et al. (1999) Z. Metallkd. 90, 1085; Richter et al. (2000) Adv. Mater. 12, 507) describe DNA as a template for metallisation in order to produce metallic nanowires. Their metallisation method involves treating DNA with an aqueous solution of Pd(CH3COO)2 for 2 hours, then adding a solution of dimethylamine borane (DMAB) as reducing agent. Palladium nanoparticles with a diameter of 3-5 nm grow on the DNA within a few seconds of the reducing agent being added. After about 1 minute, quasi-continuous coverage is achieved, with metallic aggregates being 20 nm in size.
There are two recent publications in which positively charged gold nanoparticles were attached to DNA via electrostatic assembly [Kumar et al. (2001) Advanced Materials, Vol. 13, p. 341; Sastry et al. (2001) Applied Physics Letters, Vol. 78, p. 2943]. Those papers do not discuss enlarging the attached particles via electroless plating. Richter et al. have published an article on DNA metallisation [Richter et al. (2001) Applied Physic Letters, Vol. 78, p. 536]. Also noticed was an article in which DNA was metallised by evaporation of gold [Quake and Scherer (2000) Science, Vol. 290, p. 1536] and one in which DNA was used to assemble pre-formed gold nanowires [Mbindyo et al. (2001) Advanced Materials, Vol. 13, p. 249]. None of the publications mentioned above appear to have direct bearing on the present invention. There are also several recent articles and patents in which metal nanoparticles, especially gold, are linked to oligonucleotides for diagnostic applications, but theses documents do not relate to the present invention, either.
EP 00 126 966.1 describes the metallisation of nucleic acids via metal nanoparticles produced ex-situ. This represents an improvement in the state-of-the-art methods cited above, by providing selectivity via the nucleic acid binding group(s) attached to the nanoparticles. Nevertheless, the approach has two shortcomings. One disadvantage is that it requires the synthesis of molecules capable of binding to both nucleic acids and nanoparticles (referred to as “linker molecules”).
The “in-situ” approach described in EP 00 125 823.5 circumvents many of these shortcomings, but introduces another. A disadvantage of the in-situ approach is that the density of nanoparticle nucleation sites is limited by the density of metal complexes binding to the nucleic acids in the first step of the procedure. This limitation is magnified by the fact that each particle formed during the second (reduction) step may contain tens of metal atoms. Further, although there is some control over the sites on the nucleic acids where the complexes bind, diffusion of the atoms during the second step makes it difficult to predict the location of the resulting particles.
The techniques of nucleic acid synthesis and modification have been the subject of numerous publications. In particular, these methods are described in the books Bioorganic Chemistry: Nucleic Acids (edited by S. M. Hecht, Oxford University Press, 1996) and Bioconjugate Techniques (by G. T. Hermanson, Academic Press, 1996). More specifically, the chapter by M. Van Cleve in Bioorganic Chemistry: Nucleic Acids (Chapter 3, pages 75-104) describes the techniques of “annealing” and “ligation” for assembling double-stranded nucleic acids from smaller units. The chapter by M. J. O'Donnell and L. W. McLaughlin in the same book (Chapter 8, pages 216-243) and a chapter in Bioconjugate Techniques (Chapter 17, pages 639-671) describe procedures for chemical modification of nucleic acids and oligonucleotides and the covalent attachment of reporter groups (fluorophores, spin labels, etc.). These techniques have also been used to attach metal complexes to serve as, for example, redox-active agents and catalysts for bond cleavage, but they have not been used for metallisation purposes.
An example of chemical modification is “bromine activation”. Reaction with N-bromosuccinimide, for example, causes bromination at the C-8 position of guanine residues and C-5 of cytosine. Amine nucleophiles can then be coupled to these positions by nucleophilic displacement to introduce various functional groups into nucleic acids. The sites of derivation using this method are not involved in hydrogen bonding during base pairing, so hybridization capabilities are not significantly disturbed.
“Two step” electroless plating processes are known from, for example, U.S. Pat. No. 5,503,877 and U.S. Pat. No. 5,560,960. The substrate to be plated is first exposed to a solution containing metal ion species and then to a solution of a reducing agent that reduces the metal ion species to a metal catalyst. The catalytic metal is usually Pd, but may be also at least one of Pd, Cu, Ag, Au, Ni, Pt, Ru, Rh, Os, and Ir, and is usually combined with an organic ligand containing at least one nitrogen atom. The deposited metal can be magnetic, e.g. Co, Ni, Fe and alloys, which may contain B or P introduced by the reducing agent (e.g. borohydride or hypophosphite, see U.S. Pat. No. 3,986,901; U.S. Pat. No. 4,177,253).
The gold nanoparticles prepared according to the procedure of Duff et al. are structurally related to the phosphine-stabilized M55 particles patented by Schmid [U.S. Pat. No. 4,454,070 (1984)], where M is a transition metal including Au, Rh, Ru, Pd, Ni, and Co. They are similarly related to the water-soluble, phosphine-stabilized gold particles patented by Hainfeld et al. [U.S. Pat. No. 5,360,895 (1994) and U.S. Pat. No. 5,521,289 (1996)], where the phosphine is a triphenyphosphine derivative. There are no reports of gold particles prepared according to the procedure of Duff et al. being combined with nucleic acids or using that approach for synthesizing nanoparticles of other metals or metal alloys. However, the latter should be possible, since stable water-soluble complexes of tris(hydroxymethyl) phosphine (THP) with several metals are known (e.g. Ni(0), Pd(0) Pt(0) [Ellis et al. (1992) Inorganic Chemistry, Vol. 31, p. 3026], in addition to Au(I) [Beming et al. (1998) Nuclear Medicine & Biology, Vol. 25, p. 577].
During the synthesis of the particles according to Duff et al., the following reactions are likely to occur.    (1) Neutralization of tetrakis(hydroxymethyl)phophoshphonium chloride (THPC) by sodium hydroxide generates tris(hydroxymethyl)phophine (THP) and formaldehyde:            P(CH2OH)4++OH−→P(CH2OH3)+H2CO+H2O            (2a) Reduction of hydrogen tetrachloroaurate(III) by THP in the presence of sodium hydroxide generates metallic gold and the phosphine oxide:            (2) H[AuCl4]+3 P(CH2OH3)+8 OH−→2 Au0+2 OP(CH2OH)3+Cl−+5 H2O            (2b) The formaldehyde generated in reaction (1) can also serve as the reducing agent:            4 H[AuCl4]+3 H2CO+16 OH−→4 Au0+3 CO2+16 Cl−+13 H2O            (2c) Reduction of H[AuCl4] by THP can also generate THP-gold(I) complexes, e.g.: H[AuCl4]+5 P(CH2OH)3+3 OH−→[Au{P(CH2OH)3}4]++OP(CH2OH)3+4 Cl−+2 H2O Duff et al. performed an elemental analysis of their dried preparation after dialyzing it to remove by-products and salts and found that it contained 7% P.
Aminomethyl derivatives of tris(hydroxymethyl)phosphine (THP): THP (P(CH2OH)3) is a derivative of phosphine (PH3) and formaldehyde (3×H2CO). As such, it condenses with primary (RNH2) and secondary (R1R2NH) amines in reactions related to Mannich reactions to form aminomethyl phosphine derivatives (GB Pat. 842,593 (1969); Daigle et al. (1974) Journal of Heterocyclic Chemistry, Vol. 11, p. 407; Henderson et al. (1994) Journal of the Chemical Society, Chemical Communications, p. 1863; U.S. Pat. No. 5,948,386 (1999); Berning et al. (1999) Journal of the American Chemical Society, Vol. 121, p. 1658; Krauter and Beller (2000) Tetrahedron, Vol. 56, p. 771). Like THP, complexes of aminomethyl phosphine derivatives form stable water-soluble metal complexes [Joó et al. (1996) Journal of Organometallic Chemistry, Vol. 512, p. 45; Otto et al. (1998) Inorganic Chemistry Communications, Vol. 1, p. 415; Kovács et al. (2000) Comptes Rendus de l'Académie des Sciences—Series IIC—Chemistry, Vol. 3, p. 601]. Thus, it is possible to append DNA binding agents to metal nanoparticles through such modifications. Although the THP-Au particles already exhibit DNA-binding tendencies, appended binding moieties can, for example, be used to strengthen the binding or introduce greater specificity.
Molecules containing multiple OH groups (“polyols”) such as sugars bind to DNA [Del Vecchio et al. (1999) International Journal of Biological Macromolecules, Vol. 24, p. 361; Hayashida et al (2001) Chemistry Letters, p. 277], and it has been suggested that the saccharidic residues of some antibiotics and anti-cancer drugs might function as general binding elements for DNA grooves [Nicolaou et al (1992) Journal of the American Chemical Society, Vol. 114, p. 7555]. Hydrogen bonding is probably important for such binding interactions. Certain water-soluble compounds are known to reinforce hydrogen bonding. These compounds are generally known as “kosmotropic” (i.e. structure-forming) agents or “osmolytes” [Arakawa and Timasheff (1985) Biophysical Journal, Vol. 47, p. 411; Galinski et al. (1997) Comprehensive Biochemistry and Physiology, Vol. 117A, p. 357]. Examples of kosmotropic agents are betaine, proline, and dimethylsulfoxide.